The influence of urbanization and water reclamation plants on fecal indicator bacteria and antibiotic resistance in the Los Angeles River watershed: A case study with complementary monitoring methods. - Related Documents




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307701.0000The influence of urbanization and water reclamation plants on fecal indicator bacteria and antibiotic resistance in the Los Angeles River watershed: A case study with complementary monitoring methods. Urban land use and water reclamation plants (WRPs) can impact fecal indicator bacteria (FIB) and antimicrobial resistance (AMR) in coastal watersheds. However, there is a lack of studies exploring these effects on the US West Coast. Additionally, there is limited research using a complementary approach across culture-, qPCR-, and metagenomics-based techniques for characterizing environmental AMR. In this study, sixteen locations were sampled in the Los Angeles River, encompassing both upstream and downstream of three WRPs discharging into the river. Culture-dependent methods quantified Enterococcus, total coliforms, E. coli, and extended spectrum beta-lactamase-producing E. coli as a low-cost screening tool for AMR, while qPCR measured selected antibiotic resistance genes (ARGs): sul1, ermF, tetW, blaSHV, along with intI1 and 16S rRNA genes. Bacteroides HF183 and crAssphage markers were quantified via ddPCR. All samples underwent shotgun sequencing to investigate gene abundance and mobility and an overall risk score for AMR. Results reveal downstream sites contain ARGs at least two orders of magnitude greater than upstream locations. Developed areas had the highest ARG sequence abundances and the most ARG classes as indicated by metagenomic analysis. WRP effluent exhibited elevated ARGs and co-location of ARGs, mobile genetic elements, and pathogens. A culture-based assessment of AR in E. coli and Pseudomonas aeruginosa revealed increased resistance ratios for most antibiotics from upstream to downstream a WRP discharge point. This study highlights the impacts of land use and WRPs on ARGs and FIB, offering a multi-pronged analysis of AMR.202439566612
728610.9998Influence of Seasonality and Pollution on the Presence of Antibiotic Resistance Genes and Potentially Pathogenic Bacteria in a Tropical Urban River. BACKGROUND/OBJECTIVES: This study examines how seasonality, pollution, and sample type (water and sediment) influence the presence and distribution of antibiotic resistance genes (ARGs), with a focus on antibiotic resistance genes (ARGs) located on plasmids (the complete set of plasmid-derived sequences, including ARGs) in a tropical urban river. METHODS: Samples were collected from three sites along a pollution gradient in the Virilla River, Costa Rica, during three seasonal campaigns (wet 2021, dry 2022, and wet 2022). ARGs in water and sediment were quantified by qPCR, and metagenomic sequencing was applied to analyze chromosomal and plasmid-associated resistance profiles in sediments. Tobit and linear regression models, along with multivariate ordination, were used to assess spatial and seasonal trends. RESULTS: During the wet season of 2021, the abundance of antibiotic resistance genes (ARGs) such as sul-1, intI-1, and tetA in water samples decreased significantly, likely due to dilution, while intI-1 and tetQ increased in sediments, suggesting particle-bound accumulation. In the wet season 2022, intI-1 remained low in water, qnrS increased, and sediments showed significant increases in tetQ, tetA, and qnrS, along with decreases in sul-1 and sul-2. Metagenomic analysis revealed spatial differences in plasmid-associated ARGs, with the highest abundance at the most polluted site (Site 3). Bacterial taxa also showed spatial differences, with greater plasmidome diversity and a higher representation of potential pathogens in the most contaminated site. CONCLUSIONS: Seasonality and pollution gradients jointly shape ARG dynamics in this tropical river. Plasmid-mediated resistance responds rapidly to environmental change and is enriched at polluted sites, while sediments serve as long-term reservoirs. These findings support the use of plasmid-based monitoring for antimicrobial resistance surveillance in aquatic systems.202540867992
711320.9997Stormwater loadings of antibiotic resistance genes in an urban stream. Antibiotic resistance presents a critical public health challenge and the transmission of antibiotic resistance via environmental pathways continues to gain attention. Factors driving the spread of antibiotic resistance genes (ARGs) in surface water and sources of ARGs in urban stormwater have not been well-characterized. In this study, five ARGs (sul1, sul2, tet(O), tet(W), and erm(F)) were quantified throughout the duration of three storm runoff events in an urban inland stream. Storm loads of all five ARGs were significantly greater than during equivalent background periods. Neither fecal indicator bacteria measured (E. coli or enterococci) was significantly correlated with sul1, sul2, or erm(F), regardless of whether ARG concentration was absolute or normalized to 16S rRNA levels. Both E. coli and enterococci were correlated with the tetracycline resistance genes, tet(O) and tet(W). Next-generation shotgun metagenomic sequencing was conducted to more thoroughly characterize the resistome (i.e., full complement of ARGs) and profile the occurrence of all ARGs described in current databases in storm runoff in order to inform future watershed monitoring and management. Between 37 and 121 different ARGs were detected in each stream sample, though the ARG profiles differed among storms. This study establishes that storm-driven transport of ARGs comprises a considerable fraction of overall downstream loadings and broadly characterizes the urban stormwater resistome to identify potential marker ARGs indicative of impact.201728662396
535130.9997Bacterial hosts of clinically significant beta-lactamase genes in Croatian wastewaters. Wastewater treatment plants (WWTPs) provide a suitable environment for the interaction of antibiotic resistant bacteria and antibiotic-resistance genes (ARGs) from human, animal, and environmental sources. The aim was to study the influent and effluent of two WWTPs in Croatia to identify bacterial hosts of clinically important beta-lactamase genes (blaTEM, blaVIM, blaOXA-48-like) and observe how their composition changes during the treatment process. A culture-independent epicPCR (Emulsion, Paired isolation and Concatenation Polymerase Chain Reaction) was used to identify the ARG hosts, and 16S rRNA amplicon sequencing to study the entire bacterial community. Different wastewater sources contributed to the significant differences in bacterial composition of the wastewater between the two WWTPs studied. A total of 167 genera were detected by epicPCR, with the Arcobacter genus, in which all ARGs studied were present, dominating in both WWTPs. In addition, the clinically important genera Acinetobacter and Aeromonas contained all ARGs examined. The blaOXA-48-like gene had the highest number of hosts, followed by blaVIM, while blaTEM had the narrowest host range. Based on 16S rRNA gene sequencing, ARG hosts were detected in both abundant and rare taxa. The number of hosts carrying investigated ARGs was reduced by wastewater treatment. EpicPCR provided valuable insights into the bacterial hosts of horizontally transmissible beta-lactamase genes in Croatian wastewater.202438796694
535040.9997Role of wastewater treatment plants on environmental abundance of Antimicrobial Resistance Genes in Chilean rivers. BACKGROUND: Point sources such as wastewater treatment plants (WWTPs) commonly discharge their effluent into rivers. Their waste may include antibiotic residues, disinfectants, antibiotic resistant bacteria (ARB), and Antimicrobial Resistance Genes (ARG). There is evidence that ARG can be found in the natural environment, but attribution to specific point sources is lacking. OBJECTIVES: The goal of this study was to assess the release and dissemination of ARG from three WWTPs in southern Chile via two pathways: through the river systems, and through wild birds. METHODS: A longitudinal study was conducted, collecting river sediment samples at different distances both upstream and downstream from each WWTP. Wild birds were sampled from around one of the WWTPs once a month for 13 months. A microfluidic q-PCR approach was used to quantify 48 genes covering different molecular mechanisms of resistance, and data was analyzed using ordination methods and linear mixed regression models. RESULTS: There was a statistically significant increase downstream from the WWTPs (p < 0.05) for 17 ARG, but the downstream dissemination through the rivers was not clear. Beta-lactamase genes bla(KPC), bla(TEM), and bla(SHV) were the most abundant in birds, with higher abundance of bla(SHV) in migratory species compared to resident species (p < 0.05). The gene profile was more similar between the migratory and resident bird groups compared to the WWTP gene profile. CONCLUSIONS: While results from this study indicate an influence of WWTPs on ARG abundance in the rivers, the biological significance of this increase and the extent of the WWTPs influence are unclear. In addition, wild birds were found to play a role in disseminating ARG, although association to the specific WWTP could not be ascertained.202031722832
708950.9997Poultry slaughterhouse wastewater as a driver of bacterial community shifts and the spread of antibiotic resistance genes in aquatic ecosystems. Poultry slaughterhouse wastewater (PSW) is a source of environmental pollutants, harboring pathogens and antibiotic resistance genes (ARGs). This study aimed to assess the effects of conventional biological treatment of PSW on the bacterial community and its efficiency in removing ARGs, as well as to evaluate the impact of its discharge on the receiving river. Samples were collected from raw sewage, treated effluent, and upstream and downstream river sites. Total metagenomic DNA was extracted for real-time PCR quantification of 16S rRNA, yccT gene (Escherichia coli), and ARGs, which were selected based on their ability to confer resistance to clinically relevant antibiotics and their prevalence in poultry-associated environments, including resistance to tetracyclines (tetM), beta-lactams (bla(TEM)), sulfonamides (sul1), and quinolones (qnrS). Amplicon sequencing of 16S rRNA V3-V4 region was used to assess bacterial community structure. Treated effluent significantly altered the downstream microbiome, reducing bacterial richness by up to 72.3% and diversity by 25.4%. Effluent-associated phyla such as Pseudomonadota (37%), Bacillota (28%), and Bacteroidota (26%) became dominant in the downstream river samples. Enterobacterales increased after treatment, and E. coli increased by 2.93 logs downstream. All ARGs increased after treatment and remained elevated downstream, with qnrS and sul1 rising by 3.77 and 3.87 logs, respectively. These findings highlight PSW treatment plants as a potential point of selection and dissemination of antimicrobial resistance (AMR)-related bacteria and genes. Inefficient treatment contributes to shifts in river bacterial communities and the spread of AMR.202541165913
319160.9997Profiling of Bacterial Communities of Hospital Wastewater Reveals Clinically Relevant Genera and Antimicrobial Resistance Genes. In Mexico, hospital wastewater (HWW) is a source of chemical and microbiological contamination, and it is released into the municipal sewage system without prior treatment. This water may contain pathogenic bacteria and antimicrobial resistance genes, which represent a risk to Public Health and the environment. So far, there are no studies that analyse this problem comprehensively, relating bacterial population structures, chemical contaminants, and seasonality. The aim of this work was to seasonally characterise the bacterial communities of HWW, including clinically relevant bacteria and resistance genes in Hospital Juárez de México (HJM), and to evaluate the impact of physicochemical factors on their composition. A one-year observational, cross-sectional study was conducted at five HWW discharge points of HJM. Fourteen physicochemical parameters were determined by using standard methodologies, and statistical differences between discharges and seasons were evaluated. Bacterial communities were analysed by targeted amplicon sequencing of the V3-V4 region of the 16S rRNA gene. In addition, the presence of eight antimicrobial resistance genes of local epidemiological importance was assessed. Data were analysed using alpha and beta diversity indices, principal component analysis, and multivariate statistical tests. HWW showed high taxonomic diversity, with Proteobacteria, Firmicutes, and Bacteroidetes standing out. Clinically relevant bacteria were identified in 73.3% of the analyses, with Enterobacter and Escherichia-Shigella predominating. Total and dissolved solids, temperature, nitrate, and pH significantly influenced the bacterial composition of HWW. Seven out of the eight genes evaluated were identified, with bla(KPC), bla(OXA-40), and mcr-1 being the most frequent, showing significant seasonal differences. This study underlines the microbiological and chemical complexity of HWW, highlighting the impact of clinically relevant bacteria and antimicrobial resistance genes on Public Health. The findings emphasise the need to implement hospital waste management programmes and ideally specific treatment plants to minimise the associated risks and protect the environment and human health.202540572204
728570.9997Human Activity Determines the Presence of Integron-Associated and Antibiotic Resistance Genes in Southwestern British Columbia. The dissemination of antibiotic resistant bacteria from anthropogenic sources into the environment poses an emerging public health threat. Antibiotic resistance genes (ARGs) and gene-capturing systems such as integron-associated integrase genes (intI) play a key role in alterations of microbial communities and the spread of antibiotic resistant bacteria into the environment. In order to assess the effect of anthropogenic activities on watersheds in southwestern British Columbia, the presence of putative antibiotic resistance and integrase genes was analyzed in the microbiome of agricultural, urban influenced, and protected watersheds. A metagenomics approach and high-throughput quantitative PCR (HT qPCR) were used to screen for elements of resistance including ARGs and intI. Metagenomic sequencing of bacterial genomic DNA was used to characterize the resistome of microbial communities present in watersheds over a 1-year period. There was a low prevalence of ARGs relative to the microbial population (<1%). Analysis of the metagenomic sequences detected a total of 60 elements of resistance including 46 ARGs, intI1, and groEL/intI1 genes and 12 quaternary ammonium compounds (qac) resistance genes across all watershed locations. The relative abundance and richness of ARGs was found to be highest in agriculture impacted watersheds compared to urban and protected watersheds. A downstream transport pattern was observed in the impacted watersheds (urban and agricultural) during dry months. Similar to other reports, this study found a strong association between intI1 and ARGs (e.g., sul1), an association which may be used as a proxy for anthropogenic activities. Chemical analysis of water samples for three major groups of antibiotics was below the detection limit. However, the high richness and gene copy numbers (GCNs) of ARGs in impacted sites suggest that the effects of effluents on microbial communities are occurring even at low concentrations of antimicrobials in the water column. Antibiotic resistance and integrase genes in a year-long metagenomic study showed that ARGs were driven mainly by environmental factors from anthropogenized sites in agriculture and urban watersheds. Environmental factors such as land-use and water quality parameters accounted for 45% of the variability observed in watershed locations.201829765365
711480.9997Antibiotic Resistance Genes in Freshwater Trout Farms in a Watershed in Chile. Point sources such as wastewater treatment plants, terrestrial agriculture, and aquaculture may release antibiotic residues, antibiotic resistant bacteria, and antibiotic resistance genes (ARGs) into aquatic ecosystems. However, there is a lack of quantitative studies attributing environmental ARG abundance to specific sources. The goal of this study was to evaluate the role of freshwater trout farms in the release and dissemination of ARGs into the environment. Sediment samples upstream and downstream from five rainbow trout farms were collected over time in southern Chile. A microfluidic quantitative polymerase chain reaction approach was used to quantify an ARG array covering different mechanisms of resistance, and data were analyzed using principal component analysis (PCA) and linear mixed regression models. Surveys were also conducted to obtain information about management practices, including antibiotic use, at the farms. Florfenicol and oxytetracycline were used at these farms, although at different rates. A total of 93 samples were analyzed. In the PCA, , , , , (A), (B), (C), (W), and grouped together. A statistically significant increase in abundance of , , , and several genes was found downstream from the farms compared with upstream sites, and retention ponds had the highest ARG abundance at each site. Antibiotic resistance gene levels returned to baseline at an average distance of 132.7 m downstream from the farms. Although results from this study indicate an influence of trout farms on the presence of ARGs in the immediate environment, the extent of their contribution to ARG dissemination is unknown and deserves further investigation.201931589726
307690.9997Antimicrobial resistance genes (ARGs) in sea surface aerosols over the Atlantic Ocean. The large-scale abundance and distribution of antibiotic resistance genes (ARGs) within the atmosphere remains poorly documented, particularly over oceans. This study explores bacterial loads, diversity, and associated antimicrobial resistance genes in aerosols over the North Atlantic Ocean. Aerosol samples were collected from a ship during a cruise from Brest (France) to Woods Hole (USA) for 24-h periods using a mast-mounted system, with additional one-hour spot samples taken daily and nightly using high-flow rates samplers. The airborne concentrations of bacteria along with 21 ARG subtypes as indicators of key resistance families were monitored using qPCR. These were related to the bacterial diversity obtained from same samples through ribosomal gene amplicon sequencing, and to the geographical origin of the air masses estimated using atmospheric dynamics models. Total ARG concentrations ranged from background concentrations of a few copies to >10(5) copies/m(3) of air. Near coasts, macrolide and tetracycline resistance genes were dominant (up to 93 % and 38 % of the total ARG monitored here, respectively). While sulfonamide resistance genes were also detected further offshore, those related to transposases and β-lactamases were detected only sporadically. The multiple observed correlations between the aforementioned gene concentrations in the air and potential soil-derived microorganisms may be indicative of continental inputs. Conversely, the prevalence of quinolone resistance (qepA) in the air over the open ocean points toward a contribution from marine surfaces, supported by associations between several ARGs and marine microorganisms including cyanobacteria. These may thus act as environmental reservoirs of ARGs, and sources for further environmental spread notably by air means.202541106010
3213100.9997Investigating antibiotics, antibiotic resistance genes, and microbial contaminants in groundwater in relation to the proximity of urban areas. Groundwater is an essential public and drinking water supply and its protection is a goal for global policies. Here, we investigated the presence and prevalence of antibiotic residues, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and microbial contamination in groundwater environments at various distances from urban areas. Antibiotic concentrations ranged from below detection limit to 917 ng/L, being trimethoprim, macrolide, and sulfonamide the most abundant antibiotic classes. A total of eleven ARGs (aminoglycoside, β-lactam, chloramphenicol, Macrolide-Lincosamide-Streptogramin B - MLSB, sulfonamide, and tetracycline), one antiseptic resistance gene, and two MGEs were detected by qPCR with relative abundances ranging from 6.61 × 10(-7) to 2.30 × 10(-1) copies/16S rRNA gene copies. ARGs and MGEs were widespread in the investigated groundwater environments, with increased abundances not only in urban, but also in remote areas. Distinct bacterial community profiles were observed, with a higher prevalence of Betaproteobacteria and Bacteroidetes in the less-impacted areas, and that of Firmicutes in the contaminated groundwater. The combined characteristics of increased species diversity, distinct phylogenetic composition, and the possible presence of fecal and/or pathogenic bacteria could indicate different types of contamination. Significant correlations between ARGs, MGEs and specific taxa within the groundwater bacterial community were identified, revealing the potential hosts of resistance types. Although no universal marker gene could be determined, a co-selection of int1, qacEΔ1 and sulI genes, a proxy group for anthropogenic pollution, with the tetC, tetO, tetW resistance genes was identified. As the tet group was observed to follow the pattern of environmental contamination for the groundwater samples investigated in this study, our results strongly support the proposal of this group of genes as an environmental tracer of human impact. Overall, the present study investigated several emerging contaminants in groundwater habitats that may be included in monitoring programs to enable further regulatory and protection measures.201829454283
5360110.9997Diversity, distribution and quantification of antibiotic resistance genes in goat and lamb slaughterhouse surfaces and meat products. The distribution and quantification of tetracycline, sulfonamide and beta-lactam resistance genes were assessed in slaughterhouse zones throughout meat chain production and the meat products; this study represents the first to report quantitatively monitor antibiotic resistance genes (ARG) in goat and lamb slaughterhouse using a culture independent approach, since most studies focused on individual bacterial species and their specific resistance types. Quantitative PCR (qPCR) revealed a high prevalence of tetracycline resistance genes tetA and tetB in almost all slaughterhouse zones. Sulfonamide resistance genes were largely distributed, while beta-lactam resistance genes were less predominant. Statistical analysis revealed that resistant bacteria, in most cases, were spread by the same route in almost all slaughterhouse zones, except for tetB, blaCTX and blaTEM genes, which occurred in few zones as isolated 'hot spots.' The sum of all analyzed ARG indicated that slaughterhouse surfaces and end products act as reservoirs of ARG, mainly tet genes, which were more prevalent in slaughtering room (SR), cutting room (CR) and commercial meat products (MP). Resistance gene patterns suggest they were disseminated throughout slaughterhouse zones being also detected in commercial meat products, with significant correlations between different sampling zones/end products and total resistance in SR, CR and white room (WR) zones, and also refrigerator 4 (F4) and MP were observed. Strategically controlling key zones in slaughterhouse (SR, CR and WR) by adequate disinfection methods could strategically reduce the risks of ARG transmission and minimize the issues of food safety and environment contamination.201425479100
7291120.9997Accumulation of clinically relevant antibiotic-resistance genes, bacterial load, and metals in freshwater lake sediments in Central Europe. Wastewater treatment plants (WWTP) receive the effluents from various sources (communities, industrial, and hospital effluents) and are recognized as reservoir for antibiotic-resistance genes (ARGs) that are associated with clinical pathogens. The aquatic environment is considered a hot-spot for horizontal gene transfer, and lake sediments offer the opportunity for reconstructing the pollution history and evaluating the impacts. In this context, variation with depth and time of the total bacterial load, the abundance of faecal indicator bacteria (FIB; E. coli and Enterococcus spp. (ENT)), Pseudomonas spp., and ARGs (blaTEM, blaSHV, blaCTX-M, blaNDM, and aadA) were quantified in sediment profiles of different parts of Lake Geneva using quantitative PCR. The abundance of bacterial marker genes was identified in sediments contaminated by WWTP following eutrophication of the lake. Additionally, ARGs, including the extended-spectrum ß-lactam- and aminoglycoside-resistance genes, were identified in the surface sediments. The ARG and FIB abundance strongly correlated (r ≥ 0.403, p < 0.05, n = 34) with organic matter and metal concentrations in the sediments, indicating a common and contemporary source of contamination. The contamination of sediments by untreated or partially treated effluent water can affect the quality of ecosystem. Therefore, the reduction of contaminants from the source is recommended for further improvement of water quality.201525933054
6835130.9997Metagenomic profiling of antibiotic resistance genes and their associations with the bacterial community along the Kanda River, an urban river in Japan. Antibiotic resistance genes (ARGs) present in urban rivers have the potential to disseminate antibiotic-resistant bacteria into other environments, posing significant threats to both ecological and public health. Although metagenomic analyses have been widely employed to detect ARGs in rivers, our understanding of their dynamics across different seasons in diverse watersheds remains limited. In this study, we performed a comprehensive genomic analysis of the Kanda River in Japan at 11 sites from upstream to estuary throughout the year to assess the spread of ARGs and their associations with bacterial communities. Analysis of 110 water samples using the 16S rRNA gene revealed variations in bacterial composition corresponding to seasonal changes in environmental parameters along the river. Shotgun metagenomics-based profiling of ARGs in 44 water samples indicated higher ARG abundance downstream, particularly during the summer. Weighted gene co-expression network analysis (WGCNA) linking bacterial lineages and ARGs revealed that 12 ARG subtypes co-occurred with 128 amplicon sequence variants (ASVs). WGCNA suggested potential hosts for ErmB, ErmF, ErmG, tetQ, tet (W/N/W), aadA2, and adeF, including gut-associated bacteria (e.g., Prevotella, Bacteroides, Arcobacter) and indigenous aquatic microbes (e.g., Limnohabitans and C39). In addition, Pseudarcobacter (a later synonym of Arcobater) was identified as a host for adeF, which was also confirmed by single cell genomics. This study shows that ARG distribution in urban rivers is affected by seasonal and geographical factors and demonstrates the importance of monitoring rivers using multiple types of genome sequencing, including 16S rRNA gene sequencing, metagenomics, and single cell genomics.202539488451
3217140.9997Distribution and environmental dissemination of antibiotic resistance genes in poultry farms and surrounding ecosystems. Antibiotic resistance poses a significant threat to human and animal health worldwide, with farms serving as crucial reservoirs of Antibiotic Resistance Genes (ARGs) and Antibiotic-resistant bacteria. However, the distribution of ARGs in poultry farms and their transmission patterns in the environment remain poorly understood. This study collected samples of aerosol microorganisms, cloacal matter, soil, and vegetables from poultry farms and surrounding environments at three different distances. We used 16S rRNA gene sequencing and HT-qPCR to analyze the characteristics of aerosol microbial communities and the abundance of ARGs. At the phylum level, Proteobacteria, Firmicutes, and Bacteroidetes were dominant in cloacal samples, aerosol samples, and vegetable samples, while Proteobacteria Actinobacteriota and Acidobacteria dominated soil. Pseudomonas was dominant in cloacal samples at the genus level, whereas Fusobacterium was prevalent in soil. The diversity and richness of bacterial communities were more similar between cloacal samples than those observed between either sample type compared with soil. Our results showed that tetracycline and aminoglycoside ARG relative abundance was high across all sample types but significantly increased within feces/air compared to soils/vegetables. Association analysis revealed five potential host genera for ARG/MGE presence among various microbiota populations studied here. Our findings confirm that farms are important sources for the environmental dissemination of pathogens and ARGs.202539689477
7088150.9997Small-scale wastewater treatment plants as a source of the dissemination of antibiotic resistance genes in the aquatic environment. Wastewater treatment plants (WWTPs) are significant source of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can spread further in the environment by reaching rivers together with effluents discharged from WWTPs. In this study untreated and treated wastewater (UWW, TWW), upstream and downstream river water (URW, DRW) were collected from 4 WWTPs, in the winter and autumn seasons. The occurrence of ARB resistant to beta-lactams and tetracyclines as well as the presence of antibiotics from these classes were analysed in water and wastewater samples. Additionally, the amounts of 12 ARGs, 2 genes of mobile genetic elements (MGEs), gene uidA identifying E. coli and 16S rRNA were also determined. Resistance to beta-lactams prevailed among ARB in water and wastewater samples (constituting 82-88% of total counts of bacteria). The dominant genes in water and wastewater samples were bla(TEM), tetA, sul1. The gene bla(OXA) demonstrated high variability of its concentration in samples collected in both seasons. Despite the high per cent reduction of ARB and ARGs concentration observed during the wastewater treatment processes, their large quantities are still transmitted into the environment. The research focuses on WWTPs' role in the dissemination of ARGs and MGEs in the aquatic environment.202031561123
7281160.9997City-scale monitoring of antibiotic resistance genes by digital PCR and metagenomics. BACKGROUND: Anthropogenic activities significantly contribute to the dissemination of antibiotic resistance genes (ARGs), posing a substantial threat to humankind. The development of methods that allow robust ARG surveillance is a long-standing challenge. Here, we use city-scale monitoring of ARGs by using two of the most promising cutting-edge technologies, digital PCR (dPCR) and metagenomics. METHODS: ARG hot-spots were sampled from the urban water and wastewater distribution systems. Metagenomics was used to provide a broad view of ARG relative abundance and richness in the prokaryotic and viral fractions. From the city-core ARGs in all samples, the worldwide dispersed sul2 and tetW conferring resistance to sulfonamide and tetracycline, respectively, were monitored by dPCR and metagenomics. RESULTS: The largest relative overall ARG abundance and richness were detected in the hospital wastewater and the WWTP inlet (up to ≈6,000 ARGs/Gb metagenome) with a large fraction of unclassified resistant bacteria. The abundance of ARGs in DNA and RNA contigs classified as viruses was notably lower, demonstrating a reduction of up to three orders of magnitude compared to contigs associated to prokaryotes. By metagenomics and dPCR, a similar abundance tendency of sul2 and tetW was obtained, with higher abundances in hospital wastewater and WWTP input (≈125-225 ARGs/Gb metagenome). dPCR absolute abundances were between 6,000 and 18,600 copies per ng of sewage DNA (≈10(5-7) copies/mL) and 6.8 copies/mL in seawater near the WWTP discharging point. CONCLUSIONS: dPCR was more sensitive and accurate, while metagenomics provided broader coverage of ARG detection. While desirable, a reliable correlation of dPCR absolute abundance units into metagenomic relative abundance units was not obtained here (r(2) < 0.4) suggesting methodological factors that introduce variability. Evolutionary pressure does not significantly select the targeted ARGs in natural aquatic environments.202438491508
5329170.9997Distribution of Antibiotic Resistance in a Mixed-Use Watershed and the Impact of Wastewater Treatment Plants on Antibiotic Resistance in Surface Water. The aquatic environment has been recognized as a source of antibiotic resistance (AR) that factors into the One Health approach to combat AR. To provide much needed data on AR in the environment, a comprehensive survey of antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and antibiotic residues was conducted in a mixed-use watershed and wastewater treatment plants (WWTPs) within the watershed to evaluate these contaminants in surface water. A culture-based approach was used to determine prevalence and diversity of ARB in surface water. Low levels of AR Salmonella (9.6%) and Escherichia coli (6.5%) were detected, while all Enterococcus were resistant to at least one tested antibiotic. Fewer than 20% of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (17.3%) and carbapenem-resistant Enterobacteriaceae (CRE) (7.7%) were recovered. Six ARGs were detected using qPCR, primarily the erythromycin-resistance gene, ermB. Of the 26 antibiotics measured, almost all water samples (98.7%) had detectable levels of antibiotics. Analysis of wastewater samples from three WWTPs showed that WWTPs did not completely remove AR contaminants. ARGs and antibiotics were detected in all the WWTP effluent discharges, indicating that WWTPs are the source of AR contaminants in receiving water. However, no significant difference in ARGs and antibiotics between the upstream and downstream water suggests that there are other sources of AR contamination. The widespread occurrence and abundance of medically important antibiotics, bacteria resistant to antibiotics used for human and veterinary purposes, and the genes associated with resistance to these antibiotics, may potentially pose risks to the local populations exposed to these water sources.202337998788
3192180.9997Metagenome-Wide Analysis of Rural and Urban Surface Waters and Sediments in Bangladesh Identifies Human Waste as a Driver of Antibiotic Resistance. In many low- and middle-income countries, antibiotic-resistant bacteria spread in the environment due to inadequate treatment of wastewater and the poorly regulated use of antibiotics in agri- and aquaculture. Here, we characterized the abundance and diversity of antibiotic-resistant bacteria and antibiotic resistance genes in surface waters and sediments in Bangladesh through quantitative culture of extended-spectrum beta-lactamase (ESBL)-producing coliforms and shotgun metagenomics. Samples were collected from highly urbanized settings (n = 7), rural ponds with a history of aquaculture-related antibiotic use (n = 11), and rural ponds with no history of antibiotic use (n = 6). ESBL-producing coliforms were found to be more prevalent in urban samples than in rural samples. Shotgun sequencing showed that sediment samples were dominated by the phylum Proteobacteria (on average, 73.8% of assigned reads), while in the water samples, Cyanobacteria were the predominant phylum (on average, 60.9% of assigned reads). Antibiotic resistance genes were detected in all samples, but their abundance varied 1,525-fold between sites, with the highest levels of antibiotic resistance genes being present in urban surface water samples. The abundance of antibiotic resistance genes was significantly correlated (R(2) = 0.73; P = 8.9 × 10(-15)) with the abundance of bacteria originating from the human gut, which suggests that the release of untreated sewage is a driver for the spread of environmental antibiotic resistance genes in Bangladesh, particularly in highly urbanized settings. IMPORTANCE Low- and middle-income countries (LMICs) have higher burdens of multidrug-resistant infections than high-income countries, and there is thus an urgent need to elucidate the drivers of the spread of antibiotic-resistant bacteria in LMICs. Here, we study the diversity and abundance of antibiotic resistance genes in surface water and sediments from rural and urban settings in Bangladesh. We found that urban surface waters are particularly rich in antibiotic resistance genes, with a higher number of them associated with plasmids, indicating that they are more likely to spread horizontally. The abundance of antibiotic resistance genes was strongly correlated with the abundance of bacteria that originate from the human gut, suggesting that uncontrolled release of human waste is a major driver for the spread of antibiotic resistance in the urban environment. Improvements in sanitation in LMICs may thus be a key intervention to reduce the dissemination of antibiotic-resistant bacteria.202134254820
3164190.9997Variations in Bacterial Communities and Antibiotic Resistance Genes Across Diverse Recycled and Surface Water Irrigation Sources in the Mid-Atlantic and Southwest United States: A CONSERVE Two-Year Field Study. Reduced availability of agricultural water has spurred increased interest in using recycled irrigation water for U.S. food crop production. However, there are significant knowledge gaps concerning the microbiological quality of these water sources. To address these gaps, we used 16S rRNA gene and metagenomic sequencing to characterize taxonomic and functional variations (e.g., antimicrobial resistance) in bacterial communities across diverse recycled and surface water irrigation sources. We collected 1 L water samples (n = 410) between 2016 and 2018 from the Mid-Atlantic (12 sites) and Southwest (10 sites) U.S. Samples were filtered, and DNA was extracted. The V3-V4 regions of the 16S rRNA gene were then PCR amplified and sequenced. Metagenomic sequencing was also performed to characterize antibiotic, metal, and biocide resistance genes. Bacterial alpha and beta diversities were significantly different (p < 0.001) across water types and seasons. Pathogenic bacteria, such as Salmonella enterica, Staphylococcus aureus, and Aeromonas hydrophilia were observed across sample types. The most common antibiotic resistance genes identified coded against macrolides/lincosamides/streptogramins, aminoglycosides, rifampin and elfamycins, and their read counts fluctuated across seasons. We also observed multi-metal and multi-biocide resistance across all water types. To our knowledge, this is the most comprehensive longitudinal study to date of U.S. recycled water and surface water used for irrigation. Our findings improve understanding of the potential differences in the risk of exposure to bacterial pathogens and antibiotic resistance genes originating from diverse irrigation water sources across seasons and U.S. regions.202236194536